Purified and delipidated cytochrome oxidase isolated from beef heart mitochondria is being reconstituted into single-walled lipid bilayer vesicles approximately 1500 A in diameter containing lipid mixtures of varying compositions. These reconstituted membrane particles are being characterized by freeze-etch electron microscopy; light scattering; differential scanning calorimetry; optical, laser-resonance-Raman, and nmr spectroscopy; and X-ray absorption and scattering measurements. Particular attention will be given to (i) defining the environment of the Fe and Cu sites and their charge states; (ii) mapping of the geometrical distribution of these metal atoms in the protein; and (iii) ascertaining the influence of lipid environment on the conformation of the protein and on its activity as assayed by the rate of the mediated electron transfer between ferrocytochrome-c and molecular oxygen. These studies will be conducted using bilayer vesicles containing 2-component lipid mixtures in various regions of the 2-component lipid phase diagram. Lipid mixtures with different polar headgroups, fatty acid chain lengths and extents to fatty acid unsaturation will be examined. In the course of this work, an nmr method will be developed, based on the exploitation of lipid mixtures containing both normal and deuterated phospholipids, to ascertain the structure and dynamics of mixed lipid systems and to monitor lipid-protein interactions. BIBLIOGRAPHIC REFERENCES: Effects of Structural Defects in Sonicated Phospholipid Vesicles on Fusion and Ion Premeability. Rudiger Lawaczeck, Masatsune Kainosho, Jean-Luc Girardet, and Sunney I. Chan Nature, 256, 584 (1975). Proton Magnetic Resonance Studies of Lipid Bilayer Membranes--Experimental Determination of Inter- and Intramolecular Nuclear Relaxation Rates in Sonicated Lechithin Bilayer Vesicles. Paulus A. Kroon, Masatsune Kainosho and Sunney I Chan Biochim. Biophys. Acta, 433, 282 (1976).